Reflowed gold nanostructures for surface enhanced raman spectroscopy

ABSTRACT

Methods and systems for nanopillar sensors are described. Nanopillars can be defined on a substrate, and metal deposited on the nanopillars. A thermal treatment can reflow the metal on the nanopillars forming metallic bulbs on the top end of the nanopillars. These structures can have enhanced optical detection when functionalized with biological agents, or can detect gases, particles and liquids through interaction with the metal layer on the nanopillars.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 61/938,784, filed on Feb. 12, 2014, U.S. ProvisionalPatent Application No. 62/046,628, filed on Sep. 5, 2014, U.S.Provisional Patent Application No. 62/049,256, filed on Sep. 11, 2014,and U.S. Provisional Patent Application No. 62/065,224, filed on Oct.17, 2014, and may be related to U.S. patent application Ser. No.14/621,265, titled “PLASMONICS NANOSTRUCTURES FOR MULTIPLEXINGIMPLANTABLE SENSORS” filed concurrently herewith, U.S. patentapplication Ser. No. 14/621,286, titled “REFLOWED GOLD NANOSTRUCTURESFOR SURFACE ENHANCED RAMAN SPECTROSCOPY” filed concurrently herewith,U.S. patent application Ser. No. 14/621,295, titled “SURFACE ENHANCEDRAMAN SPECTROSCOPY DETECTION OF GASES, PARTICLES AND LIQUIDS THROUGHNANOPILLAR STRUCTURES” filed concurrently herewith, and U.S. patentapplication Ser. No. 14/621,306, titled “MULTIPLEXED SURFACE ENHANCEDRAMAN SENSORS FOR EARLY DISEASE DETECTION AND IN-SITU BACTERIALMONITORING” filed concurrently herewith, the disclosures of all of whichis incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates to implantable sensors. Moreparticularly, it relates to reflowed gold nanostructures for surfaceenhanced Raman spectroscopy.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated into, and constitute apart of, this specification illustrate one or more embodiments of thepresent disclosure and, together with the description of exampleembodiments, serve to explain the principles and implementations of thedisclosure.

FIG. 1 illustrates an embodiment of a fabrication method for implantablesensors.

FIG. 2 illustrates an embodiment of a fabrication method for implantablesensors.

FIG. 3 illustrates an embodiment of a detection method for implantablesensors.

FIG. 4 illustrates an embodiment of an array of detecting regions.

FIG. 5 illustrates different types of nanopillars.

FIG. 6 illustrates an embodiment of an array of detecting regions.

FIG. 7 illustrates regions with nanopillars.

FIG. 8 illustrates reflowing of gold on the nanopillars.

FIG. 9 illustrates thiophenol measurements.

FIG. 10 illustrates TCT measurements.

FIG. 11 illustrates thrombin measurements.

FIG. 12 illustrates additional thrombin measurements.

FIG. 13 illustrates a heating test for thrombin.

FIG. 14 illustrates an aptameric assay.

FIG. 15 illustrates SERS measurements for thrombin.

FIG. 16 illustrates Raman signals for thrombin.

FIG. 17 illustrates an example of microfluidic chamber.

FIG. 18 illustrates microscopy measurements.

FIG. 19 illustrates a method to concentrate molecules.

FIG. 20 illustrates fabrication steps for a sensor.

FIG. 21 illustrates fabrication steps for nanopillars on an optic fiber.

FIG. 22 illustrates further fabrication steps for nanopillars on anoptic fiber.

FIG. 23 illustrates fabrication steps for nanopillars on an optic fiberwith a needle.

FIG. 24 illustrates measurements for thrombin experiments.

FIG. 25 illustrates different functionalization processes.

FIG. 26 illustrates an array of sensors.

FIG. 27 illustrates an example of nanopillars with bulbs.

FIG. 28 illustrates detection of hydrogen sulfide.

FIG. 29 illustrates detection of ethil-mercaptan.

FIGS. 30-31 illustrate methods of attaching nanopillars to an opticfiber.

FIGS. 32-34 illustrate data on thiophenol experiments.

SUMMARY

In a first aspect of the disclosure, a method is described, the methodcomprising: providing a substrate; defining at least one recessed regionin the substrate; forming nanopillars in the at least one recessedregion; depositing a metallic layer on a top end of the nanopillars; byheating the metallic layer, reflowing the metallic layer on the top endof the nanopillars while removing the metallic layer from a middleportion of the nanopillars, thereby forming metallic bulbs on the topend of the nanopillars; and functionalizing the metallic bulbs on thetop end of the nanopillars.

In a second aspect of the disclosure, a method is described, the methodcomprising: providing a substrate; etching at least one recessed regionin the substrate; etching at least one groove on a surface of the atleast one recessed region; forming nanopillars in the at least onerecessed region close to but not within the groove; depositing ametallic layer on a top end of the nanopillars and on a side surface ofthe at least one recessed region; by heating the metallic layer,reflowing the metallic layer on the top end of the nanopillars whileremoving the metallic layer from a middle portion of the nanopillars,thereby forming metallic bulbs on the top end of the nanopillars;functionalizing the metallic bulbs on the top end of the nanopillars;and attaching an optic fiber to the groove, the optic fiber having asurface substantially parallel and opposite to the side surface of theat least one recessed region.

In a third aspect of the disclosure, a method is described, the methodcomprising: providing a substrate; forming nanopillars on the substrate;depositing a metallic layer on a top end of the nanopillars; by heatingthe metallic layer, reflowing the metallic layer on the top end of thenanopillars while removing the metallic layer from a middle portion ofthe nanopillars, thereby forming metallic bulbs on the top end of thenanopillars; functionalizing the metallic bulbs on the top end of thenanopillars; etching the substrate on a surface opposite the surfacewith the nanopillars; inserting an optic fiber in the etched side of thesubstrate, on the surface opposite the surface with the nanopillars; andlifting the nanopillars with the optic fiber.

In a fourth aspect of the disclosure, a method to detect one or morebiomolecules is described, the method comprising: providing a sensor,the sensor comprising a substrate, at least one recessed region on thesubstrate, nanopillars defined in the at least one recessed region,metallic bulbs on a top end of the nanopillars; exposing the sensor to acomposition comprising said one or more biomolecules; and illuminating,by a laser, the metallic bulbs on the top end of the nanopillars,thereby detecting presence or absence of the one or more biomolecules.

DETAILED DESCRIPTION

The present disclosure describes several methods for fabricatingplasmonic nanostructures for implantable sensors, as well as differenttypes of implantable sensors. By initially etching into, or depositingonto, some regions of a chip it is possible to fabricate nanostructuresin recessed areas. These recessed nanostructures are less likely to bedamaged during the implantation process. The nanostructures location canbe properly controlled. The nanostructures can comprise depositedmetals, which can be subjected to a thermal treatment. By controllingthe thermal treatment, it is possible to achieve different levels ofsurface plasmon enhancement for nonlinear optical processes. Forexample, taking advantage of the nanoscopic spacing between pillars andmetal structures, it is possible to functionalize certain nanostructuresfor optical readout methods like Förster resonance energy transfer.Wavelength- or polarization-dependent extraordinary transmissions can beimplemented by varying the shapes of nanostructures to facilitateon-chip imaging of biological structures, like cells. All thesemodalities can be combined onto a single chip for multiplexingmeasurements with raster scanning of the incident laser beam. In such away, sensors can be fabricated that allow optical measurement and aresensitive to the presence of biological entities. These sensors can beimplanted in biological tissues and allow measurement of biologicalquantities. These sensors can also be implanted in a biological mediumto allow measurement of various properties, for example microorganismgrowth. Exemplary biological media will be apparent to one skilled inthe art, and include agar plates, bacterial growth media, and othersubstances.

In some embodiments, some parts of a chip containing a sensor can beleft blank in order to provide baseline signals. These blank areas canalso serve as “bar codes”. For example, regarding chip orientationduring a raster scan, the bar codes can allow the determination of thechip orientation. These blank areas can be termed as empty “grids”,while other grids, or areas, can be functionalized with known chemicalsto generate strong signals in order to identify the chip orientation. Inother words, some parts or grids of a chip may be empty, other parts maybe functionalized with known chemicals, and other parts may be used fordetection. For example, the parts used for detection can befunctionalized to detect a specific biological entity.

As known to the person skilled in the art, top-down fabrication ofplasmonics nanostructures can be carried out with a focused ion beam(FIB) technique for precise nanoscopic control of metals like Au or Ag.The FIB technique, however, can be restricted by its focusing ability,the beam tail, and the angular distribution of focused ion beams. Thesefactors can pose a limitation on the smallest achievable features, aswell as on the aspect ratio when patterning apertures on metal filmsthat are thicker than 200 nm. Moreover, the FIB method can be slow andtherefore non-scalable. Recently, Walavalkar et al. invented a newtechnique that allows scalable fabrication of plasmonicsnanostructures—see Reference [1]. Rather than being handcraftedindividually by focused ion beams, with the methods described inReference [1] plasmonics nanostructures can be produced not onlyefficiently but also with repeatability. Applications of suchnanostructures include, but are not limited to, functionalized assays,high-resolution on-chip imaging via extraordinary transmission,surface-enhanced Raman spectroscopy (SERS), and so on.

As known to the person skilled in the art, non-invasive opticalmeasurement of biophysical signals is also an important topic ofresearch. For example, monitoring of glucose levels with Ramanspectroscopy is described in Reference [2]. In a biological system,however, many biological constituents are simultaneously present, andthe signal of interest is often buried in the general background.Furthermore, for measurements that utilize a nonlinear optical effectsuch as Raman scattering, the process is typically very weak and thesignal-to-noise ratio again deteriorates. These issues can be addressedwith the implantation of a sensor that can respond optically withbiochemical specificity, or with an enhanced nonlinear optical process.

The present disclosure describes methods for fabricating implantablesensors with biochemical specificity, and with an enhanced nonlinearoptical process. In some embodiments, the methods of the presentdisclosure begin with defining regions in a silicon wafer. Other typesof materials may be used instead of silicon, when suitable for thespecific application. The person skilled in the art will understand thatvariations from the fabrication techniques described below can becarried out, when suitable for specific applications.

For example, regions to be etched in a silicon wafer can be defined withstandard techniques such as photolithography. Referring to FIG. 1, step(A) shows a cross section view of an etched wafer. Etched regions (105)are visible, together with non etched areas or mesas (110). Usingsimilar fabrication procedures known to the person skilled in the art,for example as described in Reference [1], nanoscopic patterns (115) canbe defined inside the etched regions (105). For example, techniques suchas e-beam lithography can be applied, followed by a pseudo-Bosch etch toobtain 3D-sculpted nanostructures with vertical sidewalls as shown instep (B). As can be seen from FIG. 1, the nanostructures (115) lie belowthe top surface (110), therefore there is no risk of damage during theimplantation process.

In a subsequent step, the sensor chip can be oxidized so that thenanostructures are transformed into glass, as indicated in step (C). Forexample, the silicon chip is oxidized to form a layer of silica (120).Metal can then be deposited onto the chip. For example, in step (D) gold(125) can be sputtered onto the silica. Other metals or depositiontechniques can be applied.

With an appropriate thermal treatment, the metal deposited on thenanostructures can separate from the bottom layer and wick onto thepillar tops. For example, as visible in step (E) of FIG. 1, the goldwicks on the top of the nanopillars (130) form due to the thermaltreatment. By controlling the thermal process, the spacing between themetal structures on the top (130) can be on the order of 5 to 50 nm.Such spacing presents a great improvement over the spacing that istypically achievable with a focused ion beam. Further, the whole processdescribed in the present disclosure is scalable. With such a shortspacing between metal structures, surface plasmons can greatly increasethe efficiency of Raman scattering, which facilitates opticalmeasurement through surface-enhanced Raman spectroscopy. As aconsequence, greatly improved sensors can be fabricated. The personskilled in the art will understand that different spacings are possible,for example smaller than 50 nm or greater than 5 nm.

As visible in FIG. 1, a metallic layer is deposited on the nanopillars,comprising a metallic layer on a top end of the nanopillars, themetallic layer on the top end of the nanopillars being thicker than themetallic layer on a remaining part of the nanopillars. This thicker partof the metallic layer is formed due to the application of the thermaltreatment and can have a bulbous shape.

With further fabrication steps, some of the nanopillars can befunctionalized into a chemical assay, and various methods can be usedfor optical readout of these functionalized sites. For example, Försterresonance energy transfer (FRET) can be applied to take advantage of theshort distance between the functional groups and the nearby metalstructures.

In some embodiments it is possible to remove parts of the nanopillars,obtaining a high-aspect-ratio glass region through the optically-thickmetal film. This region allows high-resolution imaging based onplasmonic extraordinary transmission for on-chip objects such as cells.In some embodiments, the sensors are optimized solely for thesehigh-aspect-ratio glass regions. An example of these regions is visiblein step (F), with a cell being detected (135). In some embodiments, thenanopillars are removed after deposition of the metallic layer (as perWalavalkar et al. in Reference [1]), therefore the region for detectionof cells comprises a non-continuous metallic layer with silicon orsilica areas where the nanopillars were previously present.

By varying the nanoscopic patterns, the transmission used for imagingmay be wavelength- or polarization-dependent, making the imagingcapability more versatile. An imaging device (145), such awirelessly-powered complementary metal-oxide semiconductor (CMOS)sensor, can be attached under the chip to transmit the images wirelesslyback to a receiver. These variations are depicted in step (F). Asvisible in step (F), some nanopillars can be functionalized (140). Forexample, antigens and antibodies can be used in the functionalization.In step (F), an example of a functionalizing agent is illustrated in ananopillar without a gold top. However, such functionalizing agents canbe applied to nanopillars with a gold top as well.

In other embodiments, other methods can be used to protect thenanostructures during the implantation process. Referring to FIG. 2,step (A), for example, an additional layer (205), such as SiO₂, can befabricated at the beginning of the fabrication process. For example, themesas (205) have been oxidized, on top of the silicon wafer. Otherregions have been etched similarly to FIG. 1. The remaining steps,(A)-(F), visible in FIG. 2 are equivalent to those in FIG. 1, apart fromthe initial oxidization step that created the oxidized regions (205).

Subsequently to fabrication, the chip can be implanted into biologicaltissues, as shown for example in FIG. 3. A laser beam (305) can beilluminated onto the implant region (310). The reflected beam (315),which may carry the Raman spectroscopic information, can be collected bythe associated optical setup. By fabricating multiple groups ofstructures on the same chip, it is possible to multiplex the measurementby raster-scanning the incident laser beam. Referring to FIG. 4, forexample, there can be different functionalized sites (405), eachfunctionalized individually for a unique biochemical target. Each site(405) can have SERS structures with different spacing between metalparts or pillar locations, so that each structure corresponds to adifferent level of enhancement. In some embodiments, there can bedifferent nanoscopic openings for wavelength- or polarization-dependentimaging on the chip. Some grids can be left blank withoutnanostructures, to serve as “bar codes” for the raster scan; in thismanner, the scan direction doesn't have to be parallel with an edge ofthe entire layout, which would be convenient when the chip is implantedin a living animal or a patient for clinical purposes. For example, whenthe laser beam scans onto the metal-coated region, which is empty ofnanostructures, the abrupt increase of reflected signals can be used toidentify the general location of the chip. The reflection from these“bar-code” areas can be used to deduce the orientation of the chip tocalculate the exact location of individual nanostructure groups for themultiplexing measurement. These blank regions can also provide abaseline signal for the ever-changing biological environment. Forexample, if the chip is implanted under the skin, reflected signals fromthese regions can serve as a baseline for the interstitial fluid andhelp resolve the enhanced signals from the nanostructures. Anothermethod to identify the chip orientation is to functionalize specificregions with known chemicals that generate strong signals. In FIG. 4 thefour corners, such as (410), are functionalized with thiol conjugated tofluorescent molecules, for example. The example of FIG. 4 can beconsidered an array of recessed regions, each with defined nanopillars,and constituting an implantable sensor.

As illustrated above, the present disclosure describes methods to etchareas on the chip, in which nanostructures are subsequently fabricated,so that they would not be damaged during the implantation process. Insome embodiments, the height of the nanopillars is equal or less thanthe height of the remaining parts of the structure, such as the mesas(110) of FIG. 1. In some embodiments, protective materials can bedeposited to protect the nanostructures from being damaged during theimplantation process. For example, oxides, metals or polymers can beused as protective materials. The protective materials can encase thesensor chip while allowing access from biological entities to the topsurface where the nanostructures are located. Due to the small spacingbetween metal structures, surface-enhanced Raman spectroscopy (SERS) canbe used for optical measurement of biochemical species on the chip. Byfunctionalizing some nanostructures, the close spacing to theneighboring metal structures allows for optical readout of thebiochemical binding. One example of a method that can be used foroptical readout of these functionalized sites is Förster resonanceenergy transfer.

Various shapes of nano structures can achieve wavelength- orpolarization-dependent extraordinary transmission, which can be used foron-chip imaging of biological structures like cells. For example, asshow in FIG. 5, the nanopillars in each region of an array of detectingregions may be parallelepipeds (505, 510), ellipsoids (515), pyramidal(520), or tapered (525), and have different lateral dimensions, height,or spacing. Different metals or different functionalizing agents may bedeposited on each detecting region in order to distinguish betweenregions. In some embodiments, each of the detecting regions may beoptimized for a different optical technique, such as SERS. The examplearray of FIG. 4 is a square array, as the array has the same number ofregions in the two lateral dimensions. FIG. 6 illustrates an example ofa rectangular array, where each area (605) is a region with nanopillars.

An imaging device can be attached to the chip, and the imaging part canbe wirelessly powered with another wireless data-link. Different typesof nanostructures can be fabricated onto the same chip for multiplexingmeasurements; the incident laser beam then raster-scans onto the chipfor the optical reading. Different functionalized sites can individuallytarget a unique biochemical species for multiplexing. Different spacingbetween pillars and/or metal structures can be fabricated to achievedifferent degrees of surface plasmon enhancement. Some regions can beleft blank without nanostructure fabrication, so that the reflectedlaser beam carries the baseline signal of its ambient environment. Theblank regions can be specifically arranged into a pattern as “bar codes”on the chip, so that the scanning results can be post-processed toidentify the chip orientation. Some regions can be functionalized withknown chemicals that generate strong optical responses to identify thechip orientation.

In some embodiments, the implantable sensors can be configured to employSERS detection. Raman spectroscopy is an inelastic scattering techniquethat can extract the spatial, chemical, conformational and bondingnature of a substance. Raman spectroscopy works by shining an incidentlaser at a known wavelength at a sample and examining the change inwavelength of the light returning from the sample, as visible forexample in FIG. 3. Changes in wavelength are caused by the absorption ofsmall amounts of energy into vibrational modes of the molecule. Theamount of energy lost to each vibration mode is unique to the type ofbond (single/double/triple), the chemical species involved in thebonding (e.g., C bonded to H), and the chemical species that surroundthe bond (e.g., whether a bond is participating in hydrogen bonding).However, this technique can be inefficient, as about 1 in 10 millionphotons participates in Raman scattering.

SERS, on the other hand, is a more efficient Raman technique. SERSrelies on the use of surface-based metallic nanostructures to enhanceand focus both the incident and outgoing light to greatly improve theefficiency of Raman scattering. One of the best structures for thisenhancement is a ‘nano-gap’ structure, featuring a nanoscale (<50 nm)gap between two electrically isolated metallic structures. An example ofsuch a structure is the nanopillars structure as illustrated for examplein FIG. 1. Light incident to a SERS structure sets up a polarizingelectric field across the gap, which is enhanced and strengthened bycharges in the metal. Previous methods known to the person skilled inthe art have relied on stochastic techniques (e.g., spinning gold beadsonto a glass substrate and hoping two are near each other), FIBtechniques, or top-down lithographic techniques to fabricate SERSstructures. The method that is presented in the present disclosure is atemplate fabrication method that combines both top-down and bottom-upfabrication to create nano-gap junctions in the nanometer range. Forexample, the spacing between nanopillars can be in the 5 nm range. TheRaman signal from these structures can be enhanced by a factor greaterthan 10^10. This technique can be used (without labels) to detect, forexample, thiophenol, tracheal cytotoxin, structures of DNA aptamers, andsingle Thrombin molecules. Tracheal cytotoxin, for example, waspreviously impossible to detect in situ or without complex HPCLtechniques, without the methods presented in the present disclosure.

Similarly to the methods described referring to FIGS. 1 and 2, SERSstructures can be fabricated on a substrate, such as a siliconsubstrate. Silicon nanopillars can be fabricated and oxidized asdescribed, for example, in U.S. Pat. No. 8,080,468, filed on Jun. 10,2010, the disclosure of which is incorporated herein by reference in itsentirety. Referring to U.S. Pat. No. 8,080,468, fabrication methods aredescribed in FIG. 1, steps a-b of U.S. Pat. No. 8,080,468. Specifically,a thin layer of titanium, followed by a 100-300 nm thick layer of gold,can be sputtered onto the substrate using an Ar+ sputtering gun (as instep c of FIG. 1 of U.S. Pat. No. 8,080,468). The chip can then beinserted in a rapid thermal annealer to cause the gold to bead on top ofthe silica nanopillars. The temperature, ramp up, and ramp downparameters can be tuned to create either spherical or rod-like shapes.These shapes can be further enhanced through other top-down andbottom-up fabrication techniques (such as, for example, ion milling,chemical etching, etc.).

Referring to FIG. 7 of the present disclosure, some examples areillustrated of the small gaps that can be created between the reflowedmetal. For example, the gap (705) between nanopillars (710, 715) coveredin a bulb of metal is smaller than the gap (720) between nanopillars notcovered by metal. As visible in FIG. 7, the nanopillars in step a) areoxidized in step b). Subsequently a metal layer is deposited in step c)and a thermal process causes the metal to bead up on top of thenanopillars as in step d). Therefore, in some embodiments, in step c)the metallic layer covers entirely the nanopillars, while after beadingup, in step d) the metallic layer covers the top end of the nanopillarsand the bottom of the regions, but not the mid portion of thenanopillars.

There can be several variations to this design with respect to the 3Dshape of the pillar (conical, sculpted, etc.), cross section of thepillar (square, rectangular, oval, etc.), cross sectional area of thepillar, spacing between pillars, initial metal thickness, reflow time,reflow temperature, cooling speed, and heating speed. Each of theseprovides a unique metallic knob or bulb in this fabrication process totune the final gold bulb diameter and gap size between gold bulbs. FIG.7 illustrates exemplary scanning electron microscope (SEM) images of anarray of nanopillars (725, 730), and nanopillars with a metallic bulb(735).

While keeping the bulbs electrically isolated from each other can beimportant for enhancing the gap-based electric field, in someembodiments it is possible to design structures that have bulbs that arenot electrically isolated yet still display Raman enhancements. Forexample, referring to FIG. 8, the SEM images (805, 810) illustrateexamples of non-electrically isolated nanopillar structures. Thephysical reason for the Raman signal of the structures of FIG. 8 comesfrom the fact that in an optical detection cycle the electrons cannotmove from one bulb to the other along the U-shaped electrical path frombulb to bulb. Therefore, for the purpose of detection, the electronsbehave in a manner that is effectively similar to that of isolatedstructures.

With regard to the functionalization of the nanopillars, a variety ofmethods can be used to attach molecules to the metallic layer. Onemethod, which can be used for example to detect Tracheal Cytotoxin, isto allow the substance, dissolved in a buffer solution, to dry on thesurface of the chip. There are a variety of other methods that can beemployed. For example, the substances of interest can be attached to thebulbs by chemical bonding, inkjet printing, metal segregation, LangmuirBlodgett films, and polymethylmethacrylate (PMMA) based maskedapproaches. One example of a chemical bonding approach is thegold-sulfur bond of a thiol group. This bond can be stronger than thegold-gold bond, as studies have shown that as the sulfur moves on themetal surface it can drag along the gold atom it is bonded to. Asillustrated in FIG. 9, using this method, it can be possible to attachthiophenol to the metallic bulbs on the nanopillars of the presentdisclosure, obtaining a SERS enhancement of 2×10⁹ when compared to theRaman spectra from thiophenol in water. One attachment method consistsin the incubation of an O₂ plasma-cleaned chip in a thiophenol solutionfor several hours. Subsequently, a rinse with DI water can be applied.The exemplary 2×10⁹ reported enhancement is the Analytical EnhancementFactor (AEF) and compares the signal received, laser spot voxel/area,and number of thiophenol molecules on the gold sphere surface/volumevoxel. By taking the ratio of the two signals (surface/volume) andaccounting for the number of molecules in the sample area of each, theenhancement factor provided by the chip can be calculated. In thisexemplary case, the AEF is an underestimation of the actual enhancementsince the field is only enhanced at most an annulus of the sphere fornon-polarized light but can be reduced to two isolated spots on eachsphere for linearly-polarized excitation (as shown from computersimulations), not over the entire surface.

An exemplary top-down fabrication method allows control of how thespacing/pillar size/annealing temperature affects the AEF. Computersimulations (905) show that the field can be enhanced by a factor of300-1000 in the hot spots of the sphere.

In some embodiments, as for example illustrated in FIG. 10, the methodsof the present disclosure can be used to find Tracheal Cytotoxin (TCT),a molecule whose spectra had not been measured previously. TCT isproduced by gram negative bacteria and destroys ciliated epithelialcells. In this exemplary method, a nanomolar concentration of TCT isallowed to dry on the substrate with metal bulb nanopillars to obtainthe Raman spectra (1005). Although there does not exist a previouslycollected Raman spectra to compare the data to, certain elements of theTCT molecule such as the bicyclic ring structure give characteristicpeaks at a Raman shift of 200 cm⁻¹ (1015) and 2100 cm⁻¹ (1010).

In some embodiments, aptamers (nucleic acid strands, for example DNA orRNA, with conformations that can bind certain biomolecules) can beattached to the surface of the gold bulbs on the nanopillars by using athiol chemistry as described above herein. Samples can be incubatedovernight in a phosphate-buffered saline (PBS) solution containing theaptamer, then rinsed several times with clean PBS solution to ensurethat there is no non-bound aptamer left on the surface. It is thenpossible to take the Raman spectra of the aptamer and see peaksassociated with its conformation. Subsequently, it is possible tosequentially heat and cool the aptamer to denature and renature it andobserve the Raman spectra change based on the conformation changes ofthe nucleic acids.

Through the methods described above, it is also possible to detectthrombin at low concentrations, for example at 500 fM concentration.Using a microfluidic chamber with an exemplary size of 1.5 cm×1.5 cm×300μm, a 500 fM concentration corresponds to 1 thrombin molecule per 15micron on a side cube. In this experimental set up, the laser spot usedfor optical measurements was 1 μm in diameter. From the concentrationstated above, detection with the laser could be taken as a keyindication of single molecule detection.

FIG. 11 illustrates a graph for the detection of thrombin. The lowercurve (1105) was taken at room temperature, with monotonicallyincreasing temperatures for the remaining curves towards the boilingtemperature (1110).

FIG. 12 illustrates the time spectra of a 500 fM thrombin solutionevolving in time. Optical heating or resistive elements can be used toheat and desorb the thrombin and then let the aptamer renature and useit for thrombin binding again. The lower curve (1205) was taken at timezero, with monotonically increasing time for the remaining curvestowards time equal to two minutes (1210).

FIG. 13 shows the heating and desorption of thrombin followed by theunraveling of the aptamer followed by stochastic conformation behaviorof the aptamer in a near boiling liquid solution. The aptamer then coolsback into shape as shown in the cooling spectra of FIG. 11. The thrombinrelated peaks are not present as the liquid was allowed to boil,denaturing the complex structure of thrombin while not allowing bindingto the aptamer. The lower curve (1305) was taken at room temperature,with monotonically increasing temperatures for the remaining curvestowards a temperature of 90 degree Celsius (1310).

FIGS. 14-16 illustrate the components and specifics of anaptamer/thrombin system, as well as specifics of the differences betweensome of the spectral peaks of the aptamer and the aptamer and thrombin.FIG. 14 illustrates exemplary aspects of aptameric assays, as well as athrombin-binding aptamer and an aptamer-thrombin structure. FIG. 15illustrates experimental measurements of thrombin-aptamer binding. FIG.16 illustrates a detailed view of the experimental Raman measurements ofthrombin shown in FIG. 15.

In some embodiments, measurements can be collected by placing the chipsin a microfluidic environment above the Raman-sensitive chip. Themicrofluidic environment can encapsulate the whole chip, with the entirechip immersed in the liquid. In other embodiments, through softlithography the microfluidics environment can be limited to an areaabove the sensing area as shown, for example, in FIG. 17. In FIG. 17,the sensor chip (1705) is within a microfluidic environment (1710). Inother embodiments, the sensing area with nanopillars (1715) is withinthe microfluidic environment (1720) while a remaining part of the chipis outside the microfluidic environment (1720). A microfluidicenvironment may be, for example, a microfluidic chamber within amicrofluidic device. In the configuration of FIG. 17 it is possible tocarry out measurements on the chip in a microscope setting. In someembodiments, the microfluidic structure may be fabricated with othermethods instead of soft lithography; for example, the microfluidicstructure may be fabricated by pressing together cover slips anddouble-sided tape.

In some embodiments, the substrate itself can be used to add a chemicalsensing mechanism to confocal microscopy as illustrated in FIG. 18. Thismethod would be useful for interrogating features on cell membranes thathave been fluorescently tagged. For example, the substrate can haveenhancement sites that match the pixel spacing of a confocal microscope.In this embodiment, the enhancement sites on the substrate arefabricated with a spacing matching the spacing of pixels in themicroscope view. This method can be used together with fluorescent tags.

In some embodiments, the sensor can be used as an optical trap, asillustrated in FIG. 19. The highly confined light in the nanogapapertures (1905) between bulbs on the nanopillars can act as a gradientelectric field to capture free-floating biological molecules (1910).This embodiment can be used to concentrate diffused molecules or targetsby passing a solution over the ‘sticky area’ multiple times while alaser (1915) is illuminating the nanostructure. Subsequently, the lasercan be turned off and the targets would diffuse away to be resuspendedin a much smaller volume of solution. Through the steps of localizationclose to nanopillars (1920) and subsequent resuspension (1925) in asmaller volume of liquid, the overall effect is to increase theconcentration of molecules in the solution. In FIG. 19, the volume ofsolution is unchanged, therefore the average concentration does notvary, although the molecules are temporarily localized in an area beforeeventually diffusing away. However, in other embodiments part of theliquid can be removed from the microfluidic environment, or the volumeof the liquid can be decreased by decreasing the size of themicrofluidic reservoir and pushing part of the liquid solution out ofthe microfluidic environment.

In other embodiments the SERS substrate can be used as a peptide orprotein sequencer. Proteins/peptides can be dragged through the electricfield maxima, similarly to the embodiment of FIG. 19, by using opticaltweezing or microfluidics techniques, while the Raman signal from thisarea is recorded. By tracking unique features of the Raman signal andcorrelating it with the position of the molecules it is possible tosequentially extract the structures that are passing through the fieldmaxima.

In some embodiments, and as illustrated in FIG. 20, optical fibers canbe integrated into the chip in a mesa-like structure, for example withthe following fabrication steps:

-   -   1) Cryo etch to define the mesa region for the sidewall        reflector;    -   2) KOH etch to define the V groove for the optical fiber;    -   3) Pseudo Bosch etch for nanopillars;    -   4) Thermal oxidation; and    -   5) Au sputtering, thermal annealing, and fiber attachment        (dotted lines indicate chip truncations with a laser scriber).

The person skilled in the art will understand that variations of theabove fabrication steps can be carried out, for example etching with adifferent agent instead of KOH, or using wet etching instead of cryoetching. An exemplary flowchart of fabrication steps is illustrated inFIG. 20, both in side view (2005) and top view (2010). A recessed regionis etched (2007), for example by cryo etching. The recessed region willhave a side mesa (2008) which acts as a reflector for the optical fiberinstalled in a subsequent step. A V groove for the optical fiber isdefined in the substrate (2009), for example with KOH. The substrate maybe made of silicon. Subsequently, the nanopillars can be defined in thesubstrate, in the recessed region (2015), with methods described abovein the present disclosure. For example, a pseudo Bosch process can beused. The top surface of the structure can then be oxidized, formingsilicon dioxide in this example (2020). A metal, for example Au, can bedeposited on the structure (2025). The optical fiber (2030) can then beattached to the structure.

In other embodiments, a remote sensing system that eliminates the chipaltogether can be fabricated as, for example, illustrated in FIG. 21.Using methods known to the person skilled in the art, a hole can be cutthrough the silicon substrate (2135). For plasmonic devices a windowfrom the front to the backside of the chip can be fabricated. Thismethod can be carried out by masking the front of the chip with apolymer, patterning a square on the back of the chip, and etchingthrough the silicon dioxide with buffered HF and through Si with XeF2,for example. As shown in FIG. 22, fiber (2240) can then be pushedthrough the hole in the chip to lift the pillars off (2245). The finaldevice is a fiber with nanopillars on the end surface (2250).

In one embodiment, the nanopillars with bulbs that act as enhancementtips are sitting within the core of the optical fiber. In thisembodiment, the nanopillars are on the illuminating end of the fiber,where the laser light exits and enters the fiber. Since, in someembodiments, the pillars are only one micron tall, they are well withinthe acceptance angle of the fiber and can be used to efficiently measureSERS signals. The fiber can work both as the pump and the signalextraction mechanism. The gold normally found on the region below thepillars can be removed chemically or by using a thinner initial metalthickness, which would wick up onto the tops of the pillars. Such adevice could have applications, for example, in remote sensing ofinorganic nitrogen for explosives detection or soil monitoring or inremote, endoscopic chemical detection within organisms. The fiber canalso be placed within a needle for blood-based or in vivo measurements,as illustrated in FIG. 23. In FIG. 23, an optical fiber with nanopillarsis attached to a needle (2310), and a close up view of the gap betweenbulbs on the nanopillars is illustrated (2305).

Devices incorporating nanopillars in implantable sensors, or sensorsthat can attach to devices which can be injected, temporarily orpermanently, can be very helpful in detecting different types ofdiseases, such as cancer and infectious diseases. Early detection ofcancer and infectious diseases can be critical to saving lives andutilizing the least invasive treatments. To detect a disease early, aclinician may look for biological markers (e.g., proteins, mRNA,cytokines, etc.) in a patient's tissues or fluids, such as blood orsaliva. Unfortunately, most diseases do not present one unique marker,but an entire constellation of molecular markers, each requiring its ownpreparation and testing for identification. The methods and devices ofthe present disclosure can provide a label-free method to detect thismarker constellation in a single test—providing unambiguous, earlyindication of a disease. One detection method that can be used, asdescribed above, is Raman spectroscopy. In Raman spectroscopy, thevibrational and chemical make-up of molecules is probed via laser light.Nanofabrication can be used to amplify the Raman signal 10 billion-foldby creating unique ‘nano-bulb’ surface enhanced Raman spectroscopy(SERS) substrates, as described above in the present disclosure. Asdescribed herein in various examples, these substrates can be sensitiveenough to track single aptamer-molecule binding events. In thefollowing, further embodiments and examples are described. Two modeldiseases used in the following are Bordetella pertussis (whooping cough)and oral cancer. Both diseases can be managed if caught early, but areoften found only once severe symptoms emerge.

In some embodiments, functionalization methods can be used to expand therange of analytes that can be detected with nano-bulb chips. Forexample, the individual binding of two oral cancer markers—interleukin-8(IL-8) protein and IL-8 coding mRNA—can be quantified individually, byfunctionalizing the gold nano-bulbs on several chips with an IL-8antibody (AB) to detect IL-8 protein and with cDNA to detect the IL-8coding mRNA. This individual quantification can be followed bymultiplexed sensitization to quantify binding of both markers,simultaneously, from a mixed sample. Furthermore, evolutionary selectioncan be used to create probes, such as aptamers or ligand probes, todetect tracheal cytotoxin (TCT), the epithelium-destroying secretion ofB. pertussis. Understanding TCT secretion from cultured B. pertussisduring growth can help decrease Pertussis-related injury to the lungs.Training in functionalization, probe design, and cell culture from theseprojects can be used to expand the scope and utility of these sensors inother applications.

In some embodiments, the methods of the present disclosure focus onmultiplexed and portable sensing. A bacterial sensor can be used, forexample, to monitor shifts in B. pertussis phenotype via proteinexpression; study of this effect can allow for targeted treatment toreduce virulence and transmissibility of whooping cough. Multiplexedfunctionalization can also be used to simultaneously detect eight keypre-oral cancer markers from a single saliva sample. In otherembodiments, SERS diagnostics can be expanded beyond the lab usinghand-held Raman spectrometers and microfluidics to create field-basedassays and fabrication to put a sensor on the tip of a fiber forin-vivo, endoscopic measurements.

The methods and devices described in the present disclosure serve as animportant early step in the long-term research goal of linkingelectro-optical sensing to the needs of diagnostic medicine. Keybiological techniques combined with nanofabrication approaches can allowfor sensor design with distinct levels of complexity for the lab,hospital, clinic, or field.

As described above, to find all markers pertaining to a disease,normally the blood has to be fractionated and different tests for eachmolecule type must be performed, delaying diagnosis. The presentdisclosure describes instead a label-free method to quantify an entireconstellation of markers in one test. The use of surface enhanced Ramanspectroscopy (SERS) provides direct chemical information about themarkers for unambiguous detection. Specifically, the sensitivity of the‘nano-bulb’ SERS substrates is exploited to make a multiplexed,lab/clinical based sensor.

For example, an early detection assay can simultaneously detect twopre-cancer markers. In a first step, the sensor can detect and quantifyIL-8 protein and IL-8 coding mRNA (both pre-cancer markers in saliva) byfunctionalizing the SERS substrate with antibodies or cDNA.Quantification can be done by calibrating relative intensities of uniqueanalyte/functionalization peaks against known concentrations. In asecond step, photo-uncageable linkers can be used to selectivelyfunctionalize half the bulbs (the metallic structures on thenanopillars) with antibody and half the bulbs with cDNA tosimultaneously detect both IL-8 protein and IL-8 mRNA in a mixedsample—a first step toward building an early disease detection device.

Additionally, it is possible to measure tracheal cytotoxin (TCT)secretion from in vitro B. pertussis (Bp) colonies. TCT secreted by Bpduring cell division is responsible for epithelial damage to the lungsin “whooping cough” and is a unique infection marker. Currently a TCTbinding molecular probe does not exist. However, it is possible tocreate an aptamer-based and peptide-based probe, using in vitroevolutionary selection methods, and use them for TCT detection. In afirst step, it is possible to make and verify binding performance ofprobes separate from the sensor. In a second step, it is possible tofunctionalize the sensor with the probes, and use the sensor to quantifyconcentrations of TCT as described above. In parallel, liquid-gellinggrowth media and an incubator can be used for culturing Bp in vitro. Byusing nanofabrication techniques the sensor can be embedded in the toplayer of agar. Subsequently, TCT release rate can be tracked in situ ascompared to the time/growth phase using the probes.

In some embodiments, in situ measurement of B. pertussis secretionsusing phenotypic markers can be carried out. A detailed study can becarried out centered on the unique 3-phase phenotypic regulatory systemof B. pertussis known as BvgAS. Based on certain environmentalconditions, the BvgAS system shifts the bacteria between dormant,infectious, and virulent phenotypes, each with a unique protein profile.The implanted sensors described in the present disclosure can be used tolook for the current phenotype through detection of the secretedproteins. Aptamers can be evolutionarily selected to bind theseproteins. Aptamers can be chosen to allow for “catch and release”measurements via optical heating so that the sensor does not becomesaturated and can remain in place during the entire experiment. A betterunderstanding of the phenotypic regulatory process of Bp can allow forearly detection and targeted treatment that can decrease virulence andtransmissibility.

In some embodiments, multiplexed detection of constellations of markersfor unambiguous disease detection can be carried out. Oral cancerpresents several non-unique proteins and mRNA fragments whosesimultaneous presence is a disease indicator. Through selectivephoto-uncaged functionalization, it is possible to create a multiplexedchip to quantify, for example, eight of these specific markersconcurrently. Development of this sensor can follow an iterative paththat leads to the creation of a new clinical early detection tool.

SERS is a powerful detection technique, but spectrometers andmicroscopes normally tether it to the lab. By using commercial,hand-held Raman devices, microfluidics, and multiplexing it is possibleto perform infrastructure-free, field-based diagnostics forconstellations of disease markers. Endoscopic sensors can be made withnanofabrication methods to place the sensor on the end of an opticalfiber. Micron sized optical fibers allow sensor placement inside 30gauge needles for in vivo analysis. These schemes extend the power ofSERS detection beyond the lab or clinic and into the world.

In some embodiments, nano-bulb SERS detection methods allow themeasurement of new bio-molecules under challenging mixed sample or invitro conditions. Early disease detection is critical to saving livesand utilizing the least invasive or irreversibly damaging treatmentprocedures. For example, finding life-threatening cancers at thepre-cancer or carcinoma in situ phase can allow for endoscopic excisionof the neoplasm before its progression requires treatment with radiationand chemotherapy. Similarly, early detection and treatment ofrespiratory bacterial infections can reduce permanent damage to theairway or lungs and reduce the duration of infectiousness to helpprevent disease transmission. In addition to improving patient health,early detection can reduce health-care costs for both hospitals andpatients through simpler treatments and preventative care. Current“detection methods” (ELISA, qPCR, etc.) are analyte specific; therefore,to find a marker pattern typical of a disease, a fluid sample must befractionated, precipitated, and tested separately for each individualtype of marker—a time and cost prohibitive method.

For example, oral cancer is a form of head and neck cancer that isindicated by cancerous growth within the mouth. Around the world morethan 125,000 people die per year as a result of this disease. InAmerica, of the approximately 37,000 people diagnosed with oral cancereach year, about two-thirds will be diagnosed once the cancer hasentered late stage III or stage IV of growth, resulting in a 62%five-year survival rate. However, if detected early and treated withlife-style changes or pre-cancer targeted excision, the prognosisimproves dramatically. To that end, there have been a great number ofstudies to determine various correlated pre-cancerous markers, but nomethod to detect these marker constellations simultaneously in aclinical setting.

As another example, whooping cough is caused by the epithelial damagedone to the respiratory tract during a Bordetella pertussis infection.Around the world, approximately 45 million people contract the diseaseand approximately 300,000 die each year, with the highest mortality ratein infants less than 4 months old. Internationally, it is the leadingcauses of vaccine preventable death. Even amongst the well-vaccinatedAmerican populace, the rate of infection is on the rise—represented byfocal outbreaks in California (in 2010), Washington and Vermont (in2012)—with the highest rate of infections since the 1950s. Earlytreatment can cut mortality, shorten the infectious period, and abaterespiratory tract damage. However, due to a partially vaccinatedpopulace and the typical delay in seeking treatment, current methods ofdetection, including qPCR and culturing, can be ineffective for earlydetection. Therefore a new, direct oral or serological detection methodis needed for early diagnosis.

As yet another example, real-time data on changes in phenotypic phase inresponse to environmental conditions can provide a unique window intothe adaptation mechanisms of a bacterial colony. For example, thebacteria B. pertussis has a unique regulatory system which transfers thebacteria between dormant, highly transmissible, and maximally toxicphenotypes. Unfortunately, the lack of animal models and the inabilityto track phenotypic specific secretions in situ has left scientists inthe dark as to what environmental conditions propel the bacteria fromphase to phase—even to the point that there is no consensus on whatcauses the “whoop” or the cough in whooping cough. The devices describedin the present disclosure can enable a method to track the bacteria'sphenotypic state in real-time, in situ and in vitro, to obtain a greaterunderstanding of the possible in vivo bacterial growth cycle to informmethods for early detection and eradication.

One detection mechanism in the present disclosure is Raman spectroscopy.In Raman spectroscopy, laser light is scattered off a molecule andreturns at a different wavelength, having donated or accepted energyfrom a vibrational mode. The wavelength differences between the incidentand collected light gives unique information about the molecule'schemical bonding, conformation and atomic species; by collecting aspectrum of these shifts, it is possible to essentially find a “chemicalfingerprint.” Unfortunately, Raman is a very weak effect, returning 1photon for every 10 million input photons, and requires long collectiontimes (minutes) and intense lasers (˜500 mW) to produce spectra.However, as described above, by creating nanostructured metallicsurfaces it is possible to amplify this effect in a technique known assurface enhanced Raman spectroscopy (SERS).

For example, the gold “nano-bulb” SERS substrate of FIG. 1 can show a10-100 billion-fold enhancement in signal over standard Ramanmeasurements. When combined with aptamer sensitization of the goldbulbs, it is possible to measure sub-clinical concentrations ofproteins, for example measuring 100 pM to 500 fM concentrations ofthrombin as illustrated in FIG. 24. The measurements represented in FIG.24 were not taken by inference, such as tracking a binding related shiftin a sensor property such as an optical resonance or threshold voltage.Instead, when the thrombin is captured by the aptamer, it is possible tosee in the SERS spectrum, unique chemical structures only found in thethrombin that was not present before binding. Furthermore, themeasurements in FIG. 24 were taken with 1 W of laser power and 5 secondcollection times; a great improvement over standard Raman sensing.

Previous Raman or SERS methods have had difficulty in performingconcentration measurements based on absolute signal intensities. Thisstems from the fact that the signal intensity of both of thesespectroscopic techniques can be heavily dependent on particular opticalexcitation and collection conditions, and SERS can also be dependent onthe location-specific enhancement factor. Therefore, particularly forSERS, it can be difficult to have an “apples to apples” comparison whencomparing concentration measurements on different chips or evendifferent locations on a single chip. In the present disclosure, amethod is described to perform “relative peak quantification,” thattakes advantage of the fabrication methods of the present disclosure tonormalize chip to chip or optical variability.

For example, quantification can be performed by first finding uniqueRaman peaks associated with (1) the molecular probe and (2) the targetmolecule. In some embodiments, these peaks were (1) the thrombin bindingaptamer (TBA) and (2) thrombin. After determining unique TBA/thrombinpeaks, solutions with known concentrations of thrombin can be applied tothe substrate and the SERS spectrum can be taken at a random location.At each thrombin concentration the same thrombin specific peak intensitycan be compared to the TBA specific peak intensity and this ratio can beplotted with respect to thrombin concentration. It is thus possible tofind that the thrombin:TBA peak intensity ratio scales linearly withthrombin concentration. Therefore, when analyzing a sample with anunknown concentration of thrombin, the concentration can be deduced byfinding the relative SERS peak ratio and locating where it sat on theconcentration curve. For example, data collected at 1-100 pMconcentrations on three separate chips is shown in FIG. 24.

Despite the random selection of measurement points and slightdifferences in enhancement factors between chips, the relative peakintensities scale identically with thrombin concentration (2405), as canbe seen in FIG. 24. This quantification method is based on two uniquefeatures. The first feature is the ability to measure both the“yardstick” and “target” peaks in a single measurement. By measuringboth the TBA and thrombin peaks in a simultaneous measurement, anyoptical measurement imperfection (misalignment, defocusing etc.) appliesequally as a transfer function to the signal from both species; scalingboth peaks but not scaling their relative intensities. By taking a 5-10s measurement any differential contributions based on probe gyration canalso be averaged out.

The second feature is that each binding site within the laser spot canbe “equal” (as in has a similar enhancement factor). Since thesubstrates used for the measurement of FIG. 24 were created throughdeterministic fabrication and not through stochastic “hot-spots”, theenhancement factor within the laser spot was uniform with respect tobinding sites. Therefore, each additional bound thrombin located in afield maximum contributed equally to an increase in the relativeintensity of its unique peak. It can be shown that this is a reasonableestimate for the substrates by raster scanning to collect the SERSsignal from a thiophenol marker and finding less than 5% variability inenhancement factor over an entire 100 μm square pad of nano-bulbs.

In FIG. 24, the top graph (2410) illustrates the evolution of 500 fMthrombin addition. Some peaks are shifted (2415) and some peaks (2420)are new thrombin specific peaks. The graph on the left (2425)illustrates the intensity ratio of thrombin, thrombin binding aptamer(TBA) vs thrombin concentration on 3 separate chips. The graph on theright (2430) illustrates optical denaturing and recovery of TBA. Guaninepeaks are blue-shifted due to reduced steric hindrance, whileconformation dependent peaks disappear during denaturing and reappearduring TBA recovery.

To extend single analyte chips to multiplexed/multi-biomoleculedetection a combination of a photo-uncageable linkers andphoto-lithographic techniques can be used. The process, an exemplaryembodiment of which is illustrated in FIG. 25, can begin by thefunctionalization of the gold nano-bulbs (2510) with athiol-alkane-amine (TAA) linker (2505). A variant of coumarin,{7-N,N-diethylamino 4-hydroxymethyl coumarin caged glutamic acid}(DECM-CG) can then be attached (2515) to the amine (2505). To preventnon-specific binding any uncomplexed TAA can be passivated with aceticanhydride (2520). Photolysis of the DECM-CG can be performed with lightat wavelengths between about 350-410 nm, conveniently capturing twocommon photo-lithographic exposure lines (365 nm and 405 nm). Aphoto-lithographic mask can be used to expose a certain area offunctionalized nano-bulbs releasing the DECM-CG, re-exposing the aminein that region (2525). A molecular probe can be attached to the aminegroup followed by acetic anhydride passivation of any left-over aminegroups. Then a new area will be uncaged by the photo-mask,functionalized and passivated, and so on, iteratively. At eachfunctionalization step, completely different binding agents, such asantibodies, aptamers (2530), cDNA, etc., can be attached creating amultiplexed/multi-molecule sensor. While this repetitive process mayseem time intensive to perform on a single-chip, using the parallelismof wafer scale lithography thousands of chips could be sensitizedsimultaneously.

As will be understood by one skilled in the art, various suitablelinkers and passivating agents may be used in the methods above. Forexample, the TAA linker may be replaced with any suitable linkercomprising an amine group, and the DECM-CG can be replaced with anysuitable photo-uncageable linker. Additionally, any appropriatepassivating agent may be used instead of the acetic anhydride.Furthermore, the above steps of creating a multiplex sensor need not beperformed on a chip as illustrated, for example, in FIG. 1 of thepresent disclosure. Any suitable substrate may be used to attach theamine-comprising linker and then the photo-uncageable linker to theamine linker. For example, a silicon or glass surface may be used in themethod of fabricating a multiplex sensor as described above.

In some embodiments, the unique reflow fabrication process described inthe present disclosure allows to make wafer scale arrays of goldnano-bulb SERS sensors with tunable enhancement gaps, for example from 5to 30 nm. In some embodiments, these structures have demonstratedaverage SERS enhancements of up to 10¹¹ over 100 μm scale pads ofpillars, and through aptamer functionalization these structures havedetected thrombin at femtomolar concentrations—4 orders of magnitudebelow clinical levels, as described above.

In some embodiments, the SERS sensors can detect two correlated oralcancer markers with single chip, multiplexed SERS. One of the oralcancer markers is Interleukin-8 (IL-8). In some embodiments, a firststep comprises functionalizing the gold nano-bulbs on several chips withan IL-8 antibody (AB). A possible gold attachment method is through ABthiolation, but other methods can be considered based on AB size andspecific residues/moieties. After functionalization, microscope-coupledSERS can be performed within a fluidic environment containingappropriate buffers to determine the unique spectral features of theantibody. Subsequently, IL-8 protein can be introduced over thesensitized chips at known sub- to supra-clinical concentrations found inpatients at risk for oral cancer and spectral features unique to IL-8residues/moieties can be determined. For each concentration, therelative intensity of IL-8 vs. AB specific peaks can be considered toperform relative peak concentration quantification as described in thepresent disclosure. In another step, the detection specificity can betested against other commonly coincident proteins due to oral cancer,such as IL-6 and IL-1. Another oral cancer marker is IL-8 coding mRNA.In some embodiments, the steps for detection of IL-8 coding mRNA aresimilar to those described above for IL-8 protein, but functionalizingwith cDNA to bind the IL-8 coding mRNA. For functionalization, thiolatedcDNA oligos can be commercially obtained; however, they are typicallyprovided as two oligos linked by a disulfide bond. Methods to cleavethis bond into two thiols, without re-oxidation, can be carried outthrough the use of biochemical tools such as reductants and de-saltingcolumns to prepare DNA for functionalization. As with the proteinmeasurements at several sub- to supra-clinical concentrations,measurements can be performed to determine the relative-peakquantification curve. To uniquely identify mRNA, one possibility is todetermine the presence of Uracil and ribose sugars in the Ramanspectrum. Specificity can be tested against other possible oral cancermRNA markers, such as mRNA coding for IL-1B and a-TNF.

Subsequently, simultaneous, multi-molecule quantification usingphoto-uncageable linkers can be carried out. In this step themultiplexed functionalization method of FIG. 25 can be used to sensitizehalf the nano-bulbs on a chip with the IL-8 AB and half with the cDNAprobe for IL-8 coding mRNA. After sensitization the chip can be placedin a buffered fluidic environment where SERS measurements can be takenof the molecular probes on each half of the nano-bulb array and comparedto previously collected spectra to check for cross contamination duringfunctionalization. Subsequently, IL-8 protein and mRNA can be introducedin a single mixed sample with the analytes at various concentrationsthat simulate baseline, pre-cancerous, and symptomatic oral cancersamples. SERS can be performed on both halves of the nano-bulbs and therelative peak intensity versus concentration results can be compared tothose previously collected to check for any confounding effects of thephotolytic functionalization. Finally, specificity can be re-verified bymeasuring the mixed sample after adding the same coincidentmRNA/proteins from above.

In other embodiments, the IL-8 antibody might not be suitable forthiolation as linkers could push the AB out of the SERS sensing zonebetween bulbs. In these embodiments, an alternative probe can be used.For example, a recently created IL-8 specific aptamer could be used as aprobe. This 44-mer hairpin can bind the IL-8 close to the bulb surfacefor effective SERS detection as seen with the TBA-thrombin system.Alternatively, custom aptamer/peptide probes can be designed asdescribed below.

Under certain conditions, nucleic acids can nonspecifically physisorbonto gold surfaces, reducing both the accuracy of concentrationmeasurements and the specificity against non-target mRNA. To preventthis, post-functionalization passivation of any empty bulb areas can beconsidered, with a mixture of short and long-chain alkane-thiols. Thismethod can create a carpet to fill in the gaps between any probes, suchas cDNA probes, and stop physisorption of nucleic acids such as mRNAdirectly onto the gold.

When uncaging the coumarin on the bulbs, UV light can become trappedbetween the chrome mask and gold substrate, exposing masked areasthrough multiple reflections. If this occurs, it is possible tosubstitute the chrome mask with an iron oxide mask which masks the chipthrough UV absorption instead of reflection and would therefore stopmultiple reflections.

To measure tracheal cytotoxin (TCT) secretion from B. pertussiscolonies, in vitro evolutionary selection of aptamer and peptide probescan be carried out. To detect TCT (the epithelial cell destroying toxinproduced during B. pertussis growth) a probe that can bind it isnecessary. To develop a TCT specific ligand, some possible methods candepend upon in vitro evolutionary selection. In these methods, a large,combinatorial library of ligands (e.g., DNA strands or peptides tetheredto their coding mRNA) is exposed to the molecular target. Any candidatesthat bind to the target are isolated and purified. The exact method ofthe isolation depends on the technique used and the target molecule. Theisolated DNA or peptide candidates are amplified (in the case of thepeptide, via the mRNA tag) and the binding/isolation process is repeatedwith this new library. After several cycles of binding andamplification, the probes with the highest affinity will bepreferentially amplified. These can be collected and sequenced; in thecase of DNA, the sequence constitutes an aptamer and in the case of themRNA the peptide translation constitutes a ligand probe. This selectioncan be performed at 37° C. to match future culturing conditions.

A next step in the above method can comprise a catching and releasingSERS detection of TCT using selected probes comprises quantifying thedetection of TCT with the two probes developed in the previous sub-aim.This step can be done with a microscope using commercially availableTCT. In a first step, the functionalization of the sensor with one ofthe probes can be carried out, subsequently taking its SERS spectrum ina buffered fluidic environment at 37° C. to find the probe'scharacteristic Raman peaks. In a next step, TCT can be introduced atvarious known concentrations and the previously mentioned relative peakintensity vs. concentration relationship can be calculated. Using thisdata it is possible to obtain an estimate of the limit of detection(LOD) of each probe when coupled to a nano-bulb SERS chip. In a nextstep, a second measurement can be taken where, with a knownconcentration of TCT in the fluidic chamber, the SERS chip is exposed toa 488 nm pulse of light to rapidly and locally heat the nano-bulbs anddenature the probe. The conformation recovery of the probe can betracked, and the return of the probe plus target signal can also betracked. This “catch and release” type aptamer recovery is similar tothat of FIG. 24 with the TBA-thrombin system. In some embodiments, achip can spend an extended period of time in a cell-culture environment;to prevent probe saturation and to find an accurate measure of thecurrent concentration of TCT, one can flash the chip and measure theconcentration immediately after recovery. The best performing probe fromthese two tests described above can be used for future ex- and in-vitrosensing.

To carry out cell culture of B. pertussis for TCT extraction andtesting, the following steps can be taken. In a first step, B. pertussiscan be cultured for TCT time correlated extraction and detection. Agarplates can be prepared, for example, with standard 15% sheep's bloodBordet-Gengou liquid setting medium. This liquid component can berendered transparent. In some embodiments, seventy plates can bestreaked with commercial B. pertussis and incubated at 37° C. for 1-14days. Each day, 5 plates can be selected and imaged to determine thestage of colony development. Subsequently, the cells/agar (fromindividual plates) can be centrifuged and the supernatant collected andpassed through a 0.2 μm filter to remove gross debris. The resultingliquid can be passed over the SERS chips sensitized with the TCT probe.The concentration of TCT in the liquid extract from each plate can bemeasured based on calibration standards developed in previous aims. Witha successful measurement it is possible to track the concentration ofTCT vs. incubation time to understand at which point during infection B.pertussis causes the most damage to respiratory epithelial cells.

In some embodiments, steric hindrance or charge neutralization at themetallic surface can cause selected probes to exhibit reduced bindingefficiency after attachment to the gold bulbs. There are some possiblesolutions to obviate this problem. For direct attachment to the gold,the thiolated end can be switched to the other side of the probe toallow for better folding without hindrance. Alternatively, athiol-alkane-amine linker can be used to tether the probe far enoughfrom the metal to allow for correct folding and to avoid chargeneutralization. Alternatively, a modified version of the selectionprocess can be performed, starting with the library of probes alreadyattached to a gold surface. Using a bead based sequestration techniqueit can be possible to segregate the binding candidates from the goldsurface and amplify them. With this method, probes can be explicitlydesigned to take into account metallic surface effects.

In the following passages, the skewed concentration that results fromcell wall polymers is discussed. TCT in a polymeric form makes up thecell wall of B. pertussis and other gram negative bacteria. In someembodiments, the biological entities of interest are the free TCTmonomers which cause epithelial cell degradation. In some embodimentsthe probes have selectivity to the TCT monomer over the polymer due tosize and conformation differences. In some cases, however, cell wallfragments may be bound. This issue may be approached with a direct orindirect approach. One example of a direct approach is to explicitly adda step in probe design that selects against cell wall polymers: oncelikely candidates have been selected with affinity to TCT it is possibleto add a TCT polymer binding step and remove the probes with affinity tothe polymer, amplifying the rest. One example of an indirect approach isto compare SERS spectra of monomer/probe vs. polymer/probe binding andfind peaks that are only present in the monomer/probe case. With thismethod, relative peak quantification can still be used to determine TCTmonomer concentration in a mixed sample.

In some embodiments, in situ measurement of B. pertussis secretions canbe carried out to understand phenotypic regulation. These embodimentsfocus on detailed study of the unique 3-phase phenotypic regulatorysystem of B. pertussis known as BvgAS. Based on environmental conditionsthe BvgAS system moves the bacteria between a dormant (Bvg−), infectious(Bvgi), and virulent (Bvg+) phenotype.

Some embodiments are directed at developing growth medium with implantedsensors. In some embodiments, the method to embed the sensor in thegrowth medium relies on the following steps. The agar plate can beprepared in two steps: first preparing and setting a standardBordet-Gengou plate, and subsequently, using a photoresist spinner, itis possible to coat a thin layer (for example about 300-500 nm) of atranslucent, blood-free B-G agar on an un-sensitized SERS chip, allowingthe bulbs to protrude from this layer. High-pressure RF oxygen plasmacan be used to clean any excess agar off the bulbs prior tofunctionalization. Subsequently, it is possible to spin a thicker layerof blood-free agar on the pre-set plate (for example, about 300 μm) andas it sets, place the coated chip in the layer. Since the chip isapproximately 350 μm thick it will protrude from the spun layer; as theagar is setting it is possible to “paint” the edges of the chip with theliquid agar so that once it sets it will be locally flush with the spunlayer. This can be done with several SERS sensors on a single plate tosimultaneously monitor several colonies.

In some embodiments, in situ measurement of TCT during B. pertussisculture can be carried out with the following methods. Using embeddedsensor plates developed in the previous steps described above combinedwith probes this method can track the emission of TCT from a singlecultured colony of B. pertussis during various stages ofincubation/growth. Prepared plates can be streaked with commercial B.pertussis taking care to ensure there is a possible culture in proximityto the embedded sensor(s). Plates can be incubated at 37° C. for 14days. At predefined intervals (for example, about 4 times a day) platescan be removed from the incubator and each time the same colony can bemeasured. Measurement can consist of a) collecting data on colony sizeand shape to estimate the stage of bacterial growth, and b) using theabove described “catch and release” SERS technique to measure the TCTconcentration at the embedded sensor. The example choice of 4measurement intervals per day is chosen based on the understanding thatB. pertussis divides at relatively slow 6 hour intervals, but can bemade more frequent as the experiment demands. This data can be comparedwith the ex-situ data collected with other methods described in thepresent disclosure, to determine the role of colony size on TCTproduction.

To develop probes for full phenotypic assay the following methods can becarried out. In the three phase BvgAS regulatory system, proteins orlack of proteins identify the current phenotype. The production of twoproteins, filamentous hemagglutinin (FHA) and Pertussis Toxin (PTx), canserve as phenotypic indicators. Absence of both proteins indicates thedormant Bvg⁻ phase; presence of PTx and FHA indicates the virulent Bvg⁺phase; and presence of FHA only indicates the highly infectious Bvg^(i)phase. Methods of amplified selection and probe development at 37° C.can be used to create aptamer and peptide probes that correspond tothese two proteins. Quantification of SERS detection, LOD determination,as well as “catch and release” performance can be completed as describedabove to determine the ideal probe for each protein that can detect lowconcentrations and survive repeated laser interrogation in cell cultureenvironments.

In some embodiments, multiplexed embedded sensor can be used to trackTCT and phenotypic phases. Methods for embedding the sensor, proximalstreaking, and multiplexed functionalization based on photo-uncaging canbe used to create agar plates with multiple, multiplexed, implantedsensors sensitized to track TCT, FHA and PTx. Following the 14-dayin-situ procedure described above it is possible to track the phenotypicphase and TCT production of individual colonies as compared to colonysize, shape, growth phase, and incubation time. Success in this aim iscritical to understanding both the developmental progress of B.pertussis infections, and the conditions under which the bacteria beginsto produce secretions that damage the epithelial tissue causingparoxysmal coughing.

Although the humidity of the incubator used to grow the bacterialcultures can be high to simulate respiratory tract conditions, thequestion remains if the water/extracellular fluid at the agar surface issufficient to ensure correct folding of molecular probes. If it is foundthat the binding affinity of the probe is reduced under theseconditions, it can be possible to add a translucent Stainer-Scholte, B.pertussis liquid growth medium on top of the plate. This medium has anionic profile similar to the buffers used in selecting the molecularprobes and is amenable to optical interrogation.

In some embodiments, the sensors described in the present disclosure canbe used to simultaneously track a peptidoglycan (TCT), a filamentousprotein (FHA), and an AB5 exotoxin (PTx) through the use of sensorsembedded in agar plates. The secretion of these molecules from the B.pertussis bacteria can serve as indicators of its infectiousness,virulence, and toxicity. With an understanding of the phenotypicevolution over time it is possible to use this same sensor system toprobe how outside influences (antibiotics, inorganic molecules, etc.)affect growth and phenotypic expression, a currently outstandingproblem. Finding a method, benign to the body, that can trap the B.pertussis bacteria in the dormant phase can allow for a reduction inrespiratory tract damage while antibiotics take their course.Additionally, by combining the multiplexing method with the methodsdescribed below, it is possible to create an early detection, rapidresponse assay to find these proteins in throat swabs or saliva. Thisassay can not only detect B. pertussis days faster than culturingmethods but, based on the protein spectrum, indicate the current phaseof infection. In other embodiments, the techniques described above forB. pertussis can be directly applied to study properties of any othercultured bacteria.

In some embodiments, multiplexed detection of constellations of markersfor unambiguous disease detection can be carried out. For example, asensor can quantify a panel of pre-cancerous oral cancer markers thatcan be used for pre-symptomatic detection in a lab or clinical setting.

In some embodiments, the following methods can be carried out to chooseand individually test eight viable pre-cancerous oral cancer markers. Ina first step, for example, eight possible markers and probes can bechosen. Some exemplary candidates can be divided by class: p proteins(IL-1, IL-6, IL-8, and TNF-a); mRNA (IL-8, IL-1p, DUSP1, OAZ1 andS100P); small RNA (miR-125, miR-200a, and miR-31); and Telomerases.

In some embodiments, four-fold multiplexed testing can be carried out.Using the photo-uncaging method described above it is possible toinitially sensitize two SERS substrates, with four molecular probes oneach, and test them against a mixed sample of the corresponding fourmarkers at known concentrations; if there is no untoward interactionsand individual marker relative peak intensity concentration curves arereplicated, it is possible to proceed to test with a mixed sample of alleight markers on both four-fold multiplexed chips.

In some embodiments, full marker panel testing under lab, blind, andsalivary conditions can be carried out. As described above, in a firststep it is possible to functionalize a SERS substrate with the eightchosen marker probes and test them against known concentrations of themarkers in a mixed solution. After verifying against knownconcentrations, it is possible to test with the researcher blindedagainst marker concentration and presence during the collection andanalysis of the relative peak data. If the experimenter in the blindedstudy can use the relative peak method to deduce the concentration ofmixed samples with greater than 90% accuracy, this device can beconsidered successful in a lab setting. Subsequently, it is possible toreplicate these results in sterile, simulated saliva and if greater than90% quantification accuracy is reported the device can be considered acandidate tool in a clinical setting.

In some embodiments, tests with human saliva can be carried out,initially using sterile saliva and spiking with known concentrations ofmarkers and then moving to use saliva samples collected by physiciansand comparing marker profiles with patient prognosis. Subsequently, thedetection can be automated rather than using manual microscopemanipulation. This can be accomplished, for example, by making opticalalignment marks to ensure the correct start point and then moving theoptical stage in a serpentine pattern to take 5-10 data points from eachdifferently sensitized area. For example, for a large 25 marker panel,with 5 data points per marker, and 5 seconds collection time, the entirechip can be read in just over 10 minutes.

In some embodiments, SERS sensors with functionalized nano-bulb chipscan be integrated with commercially available hand-held Ramanspectrometers. For example, these sensors can be used for bulk analysisof chemical samples for law enforcement or pharmaceutical applications.To compensate for the inefficiencies of Raman spectroscopy and the lackof focusing objectives these devices can be equipped with 500 mW lasers.

In some embodiments, a single chip SERS cartridge with the nano-bulbscan be integrated in a hard microfluidic chamber. For example, thesensor can comprise a microfluidic chamber as illustrated in FIG. 26.For example, a chamber (2605) can be filled with liquid and can comprisenanopillars with bulbed metal (2610). A microfluidic chamber (2615) cancomprise multiple sensors (2620). Silicon etching techniques can be usedto create a 50 micron deep chamber into which it is possible to patternthe nano-bulbs. In some embodiments, the chamber can feature input andoutput ports for loading or extracting fluid samples and be sealed witha thin glass slide for optical access as in FIG. 26. Multiplexedsensitization can be carried out prior to sealing or after sealing withthe introduction of probes through the input and output ports.

In order to create a device that can probe samples in opaque media orperform disease detection in the body, the nano-bulb sensor can beplaced on the tip of an optic fiber. In some embodiments, the goldlayer, which is normally present due to the fabrication process on theflat surface in between the nanopillars, can be removed in order to beable to etch a window in the silicon substrate and suspend thenanopillar bulbs. The resulting chip can be placed on a jig and a 50micron core, multimode fiber can be pushed through the membrane, pickingit up (with the nano-bulbs) on the end. Van der Waals forces can keepthe membrane/bulbs attached, however vapor phase glues can also be usedon the tip of the fiber prior to attachment. Multiplexed sensitizationof the bulbs can be done prior to attachment or single-probefunctionalization can occur after placement on the fiber. SERSexcitation and collection can be performed through the fiber itself.Since most of the 500 nm tall bulbs sit well within the near-fieldacceptance angle of the 50 micron fiber core this process can be quiteefficient.

By attaching the nano-bulbs to, for example, a 150 micron wide fiber theentire assembly can be threaded into a 27-30 gauge needle forintravenous measurements. Such a device could be used to monitor theblood during critical care and extract information such as clottingfactor concentration during anticoagulant administration in case of astroke or infarction.

In some embodiments, the nanopillars with a beaded metallic layerdescribed in the present disclosure can be fabricated according to thefollowing methods. For example, silicon nanopillars can be etched asdescribed above in the present disclosure, for example according toReference [4]. The pillars can be oxidized, as for example in step (C)of FIG. 1. Subsequently, gold can be sputtered on the nanopillars, forexample as in step (D) of FIG. 1. Gold can then be reflowed by heating,for example at 675° C. Gold's poor adhesion to silicon oxide combinedwith its surface tension in liquid form causes the gold to bead on topof the silicon oxide nanopillars. The nanopillars are thereforeelectrically insulated due to the absence of gold on the middle portionof the nanopillars, as visible for example in step (E) of FIG. 1. Thenanopillars are therefore electrically insulated between each other andthe substrate. By tuning the pillar spacing and gold thickness,nanometer gaps between bulbs can be uniformly formed across large areasof nanopillars. Repeatability can be ensured by use of top-downfabrication techniques to define the pillar spacing and metal thickness.A thin polymer (2705) can be spun on to passivate the remaining goldsurface (2710), as illustrated in FIG. 27. Excess polymer can be removedfrom the bulbs prior to functionalization with an oxygen plasma.

In some embodiments, the devices of the present disclosure can be usedto detect chemical compounds, for example hydrogen sulfide (H₂S).Hydrogen sulfide is a naturally occurring molecule that can be found inhigh concentrations in natural gas, crude oil, volcanos and hot springs.In these cases H₂S is formed from the hydrolysis of minerals containingsulfur. It can also be found in swamps and wells formed by the anaerobicbreakdown of organic matter by bacteria. In either case the resultingH₂S is heavier than air and tends to pool in high concentration withinlow lying areas with poor ventilation. H₂S has a wide flammability range(about 4-45%) and is quick to explode in places like mines or drillshafts. In addition H₂S is highly toxic to humans: at high levels(>500-1000 ppm) it can be almost instantly fatal; at levels above 300ppm it shuts down mitochondria and prevents cellular respiration as wellas causing pulmonary edema; above 100 ppm it shuts down the olfactorynerve preventing detection by smell and can cause irreparable eyedamage; chronic exposure to lower concentrations (<2 ppm) has beenassociated with fatigue, nausea, headaches, irritability, dizziness, andan increased risk of miscarriage.

The present disclosure describes a method for detecting H₂S at the aboveinjurious ppm range as well as in much lower concentrations such as thesingle digit ppb range. This can serve as a preventative indicator forpockets of H₂S as well as an ‘early warning’ for exposure.

The sensors for hydrogen sulfide can rely on the use of Ramanvibrational spectroscopy. In this method laser photons directed at amolecule return after donating a small fraction of their energy (andthus moving to a lower wavelength) to vibrational modes. These modes andtheir corresponding change in photon energy are unique to types ofchemical bonds and elements; thus by examining multiple shifted photonsit can be possible to determine the bonding and chemical makeup of amolecule, and therefore identify it. As such the Raman spectrum has beencalled a “chemical footprint.” As described above, standard Ramanmeasurements can be very inefficient; roughly 1 in 10 million photonsbounced off a molecule will scatter after donating energy. Thereforepowerful lasers, intricate detectors, long collection times and highconcentrations of sample molecule are normally required. Thesecharacteristics can prevent any sort of construction of a scalable andportable, ‘in-field’ Raman system that can analyze samples at ppm/ppbconcentrations.

As described in the present disclosure, by taking advantage of theunique properties of gaps between adjacent gold particles, a method canbe carried out that amplifies the Raman signal by over 1 billion. Thisis known as surface enhanced Raman spectroscopy. The SERS substrates canbe fabricated using top-down lithography as described above, obtainingelectrically isolated bulbs atop the pillars with, for example, lessthan 10 nm gaps between them.

The gold reflow process to form the bulbs, can, in some embodiments, betuned to provide gaps that are resonant at several popular wavelengthscorresponding to portable diode or gas lasers. For example, 488, 514,633, 790, and 1050 nm can be used. Other wavelengths can also be used,for example with lasers tuned deeper in the UV or Near-IR as well.

The method described herein comprises allowing the H₂S to pass over theSERS substrate, where it can form a uniquely strong Au—S bond whilereleasing a hydrogen atom resulting in an Au—S—H bonding configuration.After some time 10% of these adsorbed molecules can release the secondhydrogen resulting in an Au—S bonding configuration, as described inReference [41]. Both the S—H and the Au—S bond give unique peaks in aRaman spectrum.

FIG. 28 illustrates two spectra of the thiophenol molecule. One curve(2805) is a spectrum of a high concentration of thiophenol in solutionprior to adsorption—the peak corresponding to the S—H stretch can beeasily seen (2810) at approximately 2575 cm⁻¹. The other curve (2815) isa measurement of a single monolayer of thiophenol adsorbed on the gold.The S—H stretch peak disappears due to the donation of the H inproviding the Au—S bond. However it is possible to see peaks at 490 cm⁻¹(2820) and 1475 cm⁻¹ (2825) corresponding to the Au—S bond. The rest ofthe peaks remain similar across both spectra (2805, 2815) and correspondto modes of the carbon ring and S—C bond. In the case with the Au—S—Hcomplex formation it is possible to see both sets of peaks correspondingto Au—S and S—H with the added benefit of no other bonding features todiscriminate against. Utilizing the billion fold increase in Ramansignal associated with the SERS chip it is possible to see singlemolecules adsorbed onto the surface of the gold nanostructures—as aresult it is possible to see miniscule concentrations of H₂S adsorbedonto the surface, even at concentrations lower than the ppb range.

This method can be replicated using Cu, Al, Pt, Ni and Ag as the metalof choice, instead of Au, and is applicable to any gas, liquid, orparticulate that can be adsorbed onto the surface or that the surfacecan be immersed in. Each metal used can have a unique set of gas/liquidadsorption affinities (such as CI with AI/Ag, H₂ with Pt). In someembodiments, introduction of gas/liquid/particulate can be carried outthrough ambient air means; microchannels and/or specific molecule/atompermeable membranes can be used in the sensors; spiking the chip with aknown concentration of a calibration molecule and comparing the relativeintensities of the peaks to extract the concentration can be applied;use of two or more identical enhancement structures in proximity toserve as methods of calibration or to perform differential measurementscan be carried out; the use of on-chip light sources, detectors, andother optical components (eg., Resonators, waveguides etc.) may also beuseful; the use of the chip in coordination with a hand-held Ramanspectroscopy system is possible; the use of the chip in coordinationwith a fiber (attached in front of or on the tip of) can be carried out;the use of the chip in coordination with a microscope based Raman systemcan be carried out; the disposable use of chips may be advantageous; theintegration of the chip into a badge or portable warning/measurementsystem can be carried out; the use of functionalization ofparticles/liquid/gas with tags to aid in Raman detection can be carriedout; mass production of chips using CMOS foundries can be carried out;use of methods other than sputtering for the application of metals canbe carried out; the use of other substrates than silicon can be carriedout; the use of other methods than etching and lithography to fabricatepillars of material can be carried out; the attachment of the chip toremote probes with fiber coupling to a Raman spectrometer can be carriedout; the use of a non-regular array of structures to provide uniqueoptical effects to affect the Raman signal can be carried out; methodssuch as but not limited to oxygen plasma or H₂SO₄/H₂O₂ to clean andreuse the sensing chip can be carried out; methods of making the chipout of biocompatible materials so it can be implanted or digested can becarried out.

In the following an example is described on the detection of a gas invery low concentrations with the devices of the present disclosure. Thetest was carried out with gas used in camping applications. A typicalcomposition for camping gas is propane adulterated with 17 ppm ofethyl-mercaptan (EM). The sensing chip was exposed to the vented gasfrom a distance of a few inches. Although in the bottle the EM is at 17ppm, when vented to the air the concentration drops significantly, forexample to about 5 ppm of EM diluted in propane and air.

Samples were measured for 10 s at 50 microW of power using a 633 nmlaser. The data immediately after exposure is visible in FIG. 29 (2905)and shows a huge H—S bond-stretch signal at 2650 cm⁻¹ as well as somesmall propane related signals. This comes from physically adsorbed EMmolecules. The data in FIG. 29 is plotted at zero seconds (2905), 30 s(2910) and 60 s (2915).

When measured 30 s after exposure (2910) it was evident that some of thephysically adsorbed EM had donated a hydrogen and covalently bonded tothe gold. This gave two key peaks—one at 525 cm⁻¹ (Au—S, 2920) and oneat 2600 cm⁻¹ (S—H, 2925). Therefore, a portion of the population wasphysiabsorbed and a portion chemiabsorbed.

When measured after a minute (2915) it was found that all of thephysically adsorbed EM had covalently bonded and given up its hydrogen,leaving only an Au—S stretch signal at 525 cm⁻¹ (2930). A signal wasalso visible at 945 cm (2935) which corresponds to a C—C stretch of theethane that only becomes prominent when the molecules are aligned withan Au—S—C—C configuration perpendicular to the gold surface. The reasonis that the dipole of the vibrational mode is well aligned with thebound electric field of the laser light.

This is a first demonstration of measuring an H₂S analog gas at lowconcentrations. Based on the large signal to noise from this simplemeasurement it can be estimated that an LOD can be several orders ofmagnitude less than what measured in FIG. 29. This LOD is well past therequirements of an H₂S sensor required for oil-field applications.

In some embodiments, in order to create a “stand-off” or remote sensor,nano-bulb surface enhanced Raman substrates (SERS) can be applied ontothe ends of fibers. In the following, an embodiment of a method offabrication for the fiber-tip sensor mounting process is described. Inthis embodiment a chip was used with a triangular shaped area of SERSenhancement pillars. The triangular area is shown (3005) in FIG. 30 inan optical image (3007) and a zoomed in SEM image of the pillars (250 nmcenter to center spacing, 3010).

In the optical image (3007) the dark circle is the area of the holeetched through the silicon substrate and the T-shaped region is thelocation of the pillars. In FIG. 30, image (3015) shows the area thatwill be lifted off by the fiber. Image (3020) shows a top-down view ofthe fiber-cladding and fiber-core after lifting off the triangularregion. Image (3025) shows the side-view of the fiber after membraneattachment.

The fiber was then ashed in an oxygen plasma to remove the PMMAstabilization layer. The two SEM images (3105, 3107) in FIG. 31 show thefiber-sensor after oxygen plasma treatment. From the side view (3105) itis possible to see the layer (3110) of index matched, UV-cured epoxyused to stick the membrane to the fiber. The 45° view (3107) clearlyshows the triangular nanobulb area (3115). FIG. 31 also illustrates twocloser images (3120, 3125) of the nanobulbs after attachment. It ispossible to see that the nanopillars have remained unchanged aftertransfer to the optic fiber as described above in the presentdisclosure. The spacing, quality and density are consistent before andafter fiber mounting.

FIGS. 32-34 illustrate data on thiophenol experiments.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

The examples set forth above are provided to those of ordinary skill inthe art as a complete disclosure and description of how to make and usethe embodiments of the disclosure, and are not intended to limit thescope of what the inventor/inventors regard as their disclosure.

Modifications of the above-described modes for carrying out the methodsand systems herein disclosed that are obvious to persons of skill in theart are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which thedisclosure pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

It is to be understood that the disclosure is not limited to particularmethods or systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontent clearly dictates otherwise. The term “plurality” includes two ormore referents unless the content clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the disclosure pertains.

The present disclosure includes a References section below. Thedisclosures of all the references are incorporated by reference in theirentirety.

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What is claimed is:
 1. A method comprising: providing a substrate;defining at least one recessed region in the substrate; formingnanopillars in the at least one recessed region; depositing a metalliclayer on a top end of the nanopillars and between the nanopillars in theat least one recessed region, the metallic layer being continuousthroughout the top end of the nanopillars and between the nanopillars;by heating the metallic layer, reflowing the metallic layer on the topend of the nanopillars while removing the metallic layer from a middleportion of the nanopillars, thereby forming metallic bulbs on the topend of the nanopillars; and functionalizing the metallic bulbs on thetop end of the nanopillars.
 2. The method of claim 1, furthercomprising: defining at least one blank region in the substrate, the atleast one blank region being at least one recessed region void ofnanopillars; and depositing a metallic layer on the at least one blankregion.
 3. The method of claim 1, wherein forming nanopillars andreflowing the metallic layer comprise forming a distance between themetallic bulbs between 5 and 50 nanometers.
 4. The method of claim 1,further comprising attaching an imaging device to the substrate on asurface opposite to a surface where the nanopillars are formed.
 5. Themethod of claim 4, wherein the imaging device is a wirelessly-poweredcomplementary metal-oxide semiconductor (CMOS) device, configured totransmit images wirelessly to a receiver.
 6. The method of claim 1,wherein the substrate comprises mesas having a height higher than the atleast one recessed region on the substrate, and a height of thenanopillars is equal or less than the height of the mesas.
 7. The methodof claim 1, wherein: defining at least one recessed region comprisesdefining an array of recessed regions in the substrate, and formingnanopillars comprises forming nanopillars in each recessed region of thearray of recessed regions.
 8. The method of claim 7, wherein formingnanopillars in each recessed region of the array of recessed regionscomprises forming nanopillars with at least a first nanopillar shape inat least one recessed regions of the array of recessed regions, andforming nanopillars with at least a second nanopillar shape in at leastone recessed regions of the array of recessed regions.
 9. The method ofclaim 7, wherein functionalizing the metallic bulbs comprises using adifferent functionalizing agent for at least two regions of the array ofrecessed regions.
 10. The method of claim 7, wherein the array ofrecessed regions is a square array and each corner region of the squarearray of recessed regions is functionalized with thiol groups.
 11. Themethod of claim 1, further comprising implanting the sensor in humantissue.
 12. The method of claim 1, wherein a spacing between themetallic bulbs allows electrical insulation between the metallic bulbs.13. The method of claim 1, wherein the substrate is silicon, thenanopillars are silicon dioxide and the metallic layer is gold.
 14. Themethod of claim 1, further comprising attaching an optical fiber to oneend of the at least one recessed region, wherein an opposite wall of theat least one recessed region comprises a metallic layer, thereby actingas a reflector for the optical fiber, the optical fiber being configuredto shine a laser light on the metallic bulbs and collect a signalreflected from the metallic bulbs.
 15. The method of claim 1, furthercomprising, after forming nanopillars and prior to depositing a metalliclayer, oxidizing the nanopillars.
 16. The method of claim 1, wherein:defining at least one recessed region comprises etching the substrate;and forming nanopillars comprises etching the at least one recessedregion; and depositing a metallic layer comprises sputtering themetallic layer.
 17. The method of claim 1, further comprising applying apolymer layer on a top surface of the at least one recessed region inbetween the nanopillars.
 18. The method of claim 1, wherein formingnanopillars comprises spacing the nanopillars and the metallic bulbs ata distance based on a desired resonant wavelength.
 19. The method ofclaim 1, wherein heating the metallic layer is at 675° C.
 20. The methodof claim 1, further comprising, after defining at least one recessedregion and prior to forming nanopillars, forming mesas in the substrateand oxidizing the mesas.
 21. The method of claim 1, whereinfunctionalizing the metallic bulbs comprises applying one or morefunctionalizing agents on the metallic bulbs on the top end of thenanopillars.
 22. The method of claim 21, wherein the one or morefunctionalizing agents is a nucleic acid aptamer.
 23. The method ofclaim 21, wherein the one or more functionalizing agents comprise anantibody.
 24. The method of claim 21, wherein the one or morefunctionalizing agents comprise cDNA.
 25. The method of claim 21,wherein the one or more functionalizing agents comprise two or moredifferent functionalizing agents.
 26. The method of claim 21, whereinthe one or more functionalizing agents specifically bind to one or morebiomolecules.
 27. The method of claim 26, wherein said binding isreversible.
 28. The method of claim 26, wherein at least one of the oneor more biomolecules is a protein.
 29. The method of claim 26, whereinat least one of the one or more biomolecules is an mRNA.
 30. The methodof claim 26, wherein at least one of the one or more biomolecules is acytokine.
 31. The method of claim 26, wherein the one or morebiomolecules comprise a molecular marker of disease.
 32. The method ofclaim 31, wherein the disease is oral cancer.
 33. The method of claim31, wherein the disease is whooping cough.
 34. The method of claim 26,wherein the one or more biomolecules comprise tracheal cytotoxin. 35.The method of claim 26, wherein the one or more biomolecules comprisethrombin.
 36. A method comprising: providing a substrate; etching atleast one recessed region in the substrate; etching at least one grooveon a surface of the at least one recessed region; forming nanopillars inthe at least one recessed region close to but not within the groove;depositing a metallic layer on a top end of the nanopillars, on a sidesurface of the at least one recessed region, and between the nanopillarsin the at least one recessed region, the metallic layer being continuousthroughout the top end of the nanopillars and between the nanopillars;by heating the metallic layer, reflowing the metallic layer on the topend of the nanopillars while removing the metallic layer from a middleportion of the nanopillars, thereby forming metallic bulbs on the topend of the nanopillars; functionalizing the metallic bulbs on the topend of the nanopillars; and attaching an optic fiber to the groove, theoptic fiber having a surface substantially parallel and opposite to theside surface of the at least one recessed region.
 37. The method ofclaim 36, wherein heating the metallic layer is at 675° C.
 38. A methodcomprising: providing a substrate; forming nanopillars on the substrate;depositing a metallic layer on a top end of the nanopillars and betweenthe nanopillars in the at least one recessed region, the metallic layerbeing continuous throughout the top end of the nanopillars and betweenthe nanopillars; by heating the metallic layer, reflowing the metalliclayer on the top end of the nanopillars while removing the metalliclayer from a middle portion of the nanopillars, thereby forming metallicbulbs on the top end of the nanopillars; functionalizing the metallicbulbs on the top end of the nanopillars; etching the substrate on asurface opposite the surface with the nanopillars; inserting an opticfiber in the etched side of the substrate, on the surface opposite thesurface with the nanopillars; and lifting the nanopillars with the opticfiber.
 39. The method of claim 38, further comprising inserting theoptic fiber with the nanopillars within a needle.
 40. The method ofclaim 38, further comprising applying a layer of glue to a surface ofthe optic fiber prior to inserting, thereby gluing the nanopillars tothe optic fiber when lifting the nanopillars with the optic fiber. 41.The method of claim 38, wherein heating the metallic layer is at 675° C.